Glioblastoma (GBM) is among the most lethal human cancers, but despite decades of extensive effort, median survivals remain one year, or less. Because invasion and stemness are inter-connected properties that drive much of glioma malignancy, genes that co-regulate these phenotypes are robust therapeutic targets. One such candidate is TWIST1 (TW), a bHLH transcription factor (TF) central to epithelial mesenchymal transition (EMT), invasion and stemness and metastasis in carcinomas and normal development. In gliomas TW is upregulated in higher grade tumors and promotes GBM cell invasion and stemness in vitro and viability and growth of glioma stem cells (GSCs) in vivo. These findings strongly support its potential as a therapeutic target for GBM but identifying drug inhibitors of TFs has proven difficult by traditional means. The purpose of this proposal then is t develop indirect methods for targeting TW in GSCs through downstream genes essential to its malignant activity. TW must form a homo or heterodimers with other bHLH proteins to regulate gene transcription and the specific dimer motif defines unique phenotypes and gene expression sub-networks. The activation of unique gene expression patterns, or sub-networks, specific TW dimer partners which also map to specific malignant phenotypes will be leveraged to identify targets critical for TW activation of GSC invasion and survival. Further, phosphorylation of TW regulates dimer partner affinities and invasive behavior of GBM cells. Based on these observations this proposal will test the hypothesis that TW dimer specific pathways (sub-networks) can identify therapeutic targets to inhibit TW-regulated malignancy. AIM 1 employs proteomics to profile TW bHLH family binding partners associated with increased or decreased invasion and stemness. To facilitate this screen differential invasion and dimer affinity will be introduced through expression of pro- invasive wild-type and an anti-invasive TW phospho-mutant. In AIM2 candidate dimers validated to regulate TW binding and invasive or stem-like phenotypes will be tested to establish their impact on tumorigenicity and malignant phenotypes (invasion, proliferation, survival and angiogenesis) in vivo. In AIM3 gene expression arrays and ChIP-seq will define TW dimer specific sub-networks and identify direct and indirectly regulated TW targets. Candidate targets rank-ordered by comprehensive bioinformatic analysis of GO functional categories, pathways and expression in public GBM databases will then be tested in vitro and in vivo to validate equivalency with TW inhibition in selected GSC lines. This innovative approach is expected to identify TW dimer specific gene expression sub-networks and targets that functionally phenocopy the effects of TW inhibition on invasion, GSC tumorigenicity and glioma malignancy. This strategy is expected to have therapeutic relevance that translates to other TFs currently considered undruggable. Of significance, these studies will provide new mechanistic insight with broad implications for understanding the role of TW in development, EMT and the many other cancers in which TW promotes malignancy.